Rapid and simple quantification of belimumab in human plasma using ultra-high performance liquid chromatography with...

Belimumab is a monoclonal antibody against the B-lymphocyte stimulating factor and is approved for the treatment of patients with systemic lupus erythematosus (SLE) not responding adequately to existing therapies. In this study, we established and validated an assay for quantifying belimumab in human plasma.

From the peptides generated by trypsin digestion of belimumab, in silico analysis was used to search for unique peptides to determine the surrogate peptides. Samples were trypsin digested, pretreated with solid phase extraction, and analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) to quantify the surrogate peptide in the samples. The assay was validated according to the Food and Drug Administration (FDA) bioanalytical method validation guidance. We used the established assay to quantify plasma belimumab concentrations in two SLE patients treated with belimumab.

Among the unique peptides identified by the in silico analysis, the peptide with the best peak shape when measured by UHPLC-MS/MS was selected as the surrogate peptide. The validation results of this assay met the acceptable criteria recommended by the FDA guidance. The lower limit of quantification (LLOQ) for belimumab was 2 µg/mL. Recovery rates and matrix effects when corrected for internal standards were 91.5-114.3 % and 96.9-108.4 %, respectively. Plasma concentrations of belimumab were measured in 12 samples from two belimumab-treated SLE patients. All concentrations were within the calibration range.

We have established and validated a method for measuring plasma belimumab concentrations using UHPLC/MS-MS. By measuring plasma belimumab concentrations in more patients, this method is expected to contribute to appropriate use of belimumab.